Xanthomonas campestris ATCC 31600 and process for use

ABSTRACT

A new degenerative-resistant strain of Xanthomonas campestris is described which is suitable for longterm continuous fermentation to produce xanthan gum from a nutrient medium, typically containing inorganic salts, glucose, NH 4  Cl, and citric acid, with or without yeast extract or yeast autolysate.

INTRODUCTION

The present invention relates to the production of heteropolysaccharidesby the action of certain novel degenerative resistant strains ofXanthomonas campestris on minimal media. More particularly, it isconcerned with the production of xanthan gum by the use of these novelbacteria on aqueous nutrient media having assimilable sources of carbon,nitrogen and inorganic substances.

BACKGROUND

Batch fermentation of inoculated medium with Xanthomonas campestris NRRLB-1459 for 36-72 hours under aerobic conditions results in the formationof xanthan gum, which is separated from the other components of themedium by precipitation with acetone or methanol in a known manner.Because of time required to ferment each batch, the low biopolymercontent of the fermented medium and the processing required for therecovery and purification of the product, xanthan gum produced by batchfermentation, hereinafter also referred to as xanthan, is relativelyexpensive.

Because continuous operation of a fermentation process offers a numberof potential advantages over conventional batch methods that could bereflected in lower costs, considerable effort has been put forth in thepast to perfect conditions that would support a reliable continuousprocess. But even with a continuous process a cheap medium from whichxanthan can be produced is required. In addition to the necessity of aninexpensive medium in the manufacture of a low cost xanthan product, theratio of xanthan to cells (bacteria) should be as high as possible inorder to reduce subsequent filtration costs for cell removal. Thespecific productivity of the culture employed also should be as high aspossible in order to maintain the aforesaid high ratio as well as toreduce vessel volume and capital costs. The expression "specificproductivity" as used in the present description is intended to mean thenumber of grams of xanthan produced/grams of cells/hour. The cultureshould be stable under continuous culture conditions on a long termbasis to avoid frequent restarts and lost productivity.

Although xanthan has been produced by continuous fermentation in thepast, such methods have not met with unqualified success. In some cases,vitamins and/or amino acids had to be employed in the media insubstantial quantities in order to avoid culture degeneration or toimprove specific productivity. Use of these additives, as well assoybean protein, cotton seed protein, etc., all tend to make the xanthanthus produced more costly.

It is well known that the continuous production of xanthan has beenhampered by a tendency of the culture Xanthomonas campestris B-1459 tochange or degenerate after a fairly small and specific number ofturnovers, i.e., the time required during the fermentation to completelyreplace one volume of broth in the fermentation vessel. Normally, 6-9turnovers are the maximum that can be obtained before degeneration ofthe culture occurs. At the same time, there is a decrease in viscosity,a loss in volumetric productivity of xanthan, i.e., grams ofxanthan/liter of broth/hour, and appearance of a variety of culturevariants or strains that no longer produce xanthan or else produce axanthan of low quality. It has been demonstrated, for example, thatculture degeneration occurs when dried distillers solubles (DDS) is usedin the medium as the complex nitrogen source, whether in the whole formor as a water soluble extract. In other cases, certain strains ofXanthomonas have been grown successfully without culture degeneration insimple minimal media, but the xanthan:cell ratio and specificproductivity have been low, on the order of 0.1-0.12 gm xanthan/gmcells/hr.

Earlier work has indicated that heteropolysaccharides produced by theaction of Xanthomonas bacteria on carbohydrate media have potentialapplications as film forming agents, as thickeners for body buildingagents in edible products, cosmetic preparations, pharmaceuticalvehicles, oil field drilling fluids, fracturing liquids and similarcompositions and as emulsifying, stabilizing and sizing agents.Heteropolysaccharides, particularly xanthan gum, have significantpotential as a mobility control agent in micellar polymer flooding. Thisgum has excellent viscosifying properties at low concentration, isresistant to shear degradation and exhibits only minimal losses inviscosity as a function of temperature, pH and ionic strength. For thesereasons, xanthan gum is an attractive alternative to syntheticpolyacrylamides for enhanced oil recovery operations.

SUMMARY OF THE INVENTION

We have now discovered a degenerative-resistant strain of Xanthomonascampestris and have developed a process for using this strain whicheffectively overcomes the problems of xanthan production recited above.This strain of Xanthomonas campestris which we have designatedXanthomonas campestris XCP-1 ATCC 31600 is capable of continuouslyproducing xanthan at high specific productivities, i.e., 0.24 to 0.32 gmxanthan/gm cells/hr, for several hundred hours without culturedegeneration from inexpensive aqueous nutrient media such as, forexample, minimal medium consisting primarily of inorganic salts, glucoseand NH₄ Cl. The medium may or may not also contain a yeast extract oryeast autolysate as a supplemental nitrogen source. Generally, it may besaid that any medium having assimilable sources of carbon, nitrogen andinorganic substances will serve satisfactorily for use with this neworganism.

The process of our invention in which this new strain is utilized can beeither a single stage or two-stage continuous fermentation process. Inthe single stage embodiment the organism is grown, preferably underconditions such that the quantity of one of the growth nutrients presentis limited. The quantity of biomass obtained will be determined by theconcentration of the limiting nutrient. A portion of the residualglucose or equivalent sugar present is converted to xanthan gum and thelatter ultimately recovered from the fermentation effluent. In thetwo-stage process, the aforesaid fermenter effluent is taken to a secondfermentation stage where additional glucose or equivalent sugar isintroduced and converted to xanthan. In operation of the second stage, abalance of the flow of the first stage effluent and glucose solutionmust approximate the flow rate of the second stage effluent. The growthlimiting nutrients normally employed are nitrogen, phosphorous orsulfur.

SPECIFIC EMBODIMENTS OF THE INVENTION

Subcultures of this living organism can be obtained upon request fromthe permanent collection of the American Type Culture Collection, 12301Parklawn Drive, Rockville, MD 20852. The accession number in thisrepository for Xanthomonas campestris XCP-1 is given above. The novelXanthomonas campestris strain referred to was isolated from a pilotplant run in which a 28 liter fermenter was operated using a culture ofXanthomonas campestris NRRL B1459, growing in a suitable nutrient mediumhaving a composition shown in Table I.

                  TABLE I                                                         ______________________________________                                        Component        Concentration (ppm)                                          ______________________________________                                        Glucose          22,000                                                       NH.sub.4 Cl      300 as N                                                     KOH              1000 + as K                                                  H.sub.3 PO.sub.4 300 as P                                                     MgSO.sub.4       100 as Mg                                                    CaCl.sub.2       10 as Ca                                                     NaCl             10 as Na                                                     FeSO.sub.4       2 as Fe                                                      ZnSO.sub.4       0.35 as Zn                                                   MnSO.sub.4       0.35 as Mn                                                   Na.sub.2 MoO.sub.4                                                                             0.21 as Mo                                                   H.sub.3 BO.sub.3 0.07 as B                                                    KI               0.14 as I                                                    CuSO.sub.4       0.020 as Cu                                                  Citric Acid      250                                                          ______________________________________                                    

Continuous fermentation of the above medium was initially conducted at28° C., a pH of 6.8, an aeration rate of 0.2-0.4 vol./vol./min.,agitation rate of 230 rpm, dissolved oxygen of 90% saturation, and adilution rate of 0.08 hr⁻¹. After about 6 turnovers, the culture beganto degenerate with lowered xanthan productivity and viscosity. At thispoint, several changes were made to the medium. The pH was lowered to6.4 from 6.8; iron, copper and zinc concentrations in the broth wereraised to 3, 0.6 and 1 ppm, respectively, by adding these trace elementsto the glucose feed tank (they were previously added with the rest ofthe inorganic salts), the citric acid level was doubled to 500 ppm, andH₃ PO₄ and MgSO₄ levels were reduced by half. For 2 to 3 cultureturnovers after these changes the culture continued to degenerate withviscosity dropping to 70 cp and specific productivity decreasing to 0.7gm/gm cells/hr.

During an overnight period, the exit line, from the fermenter pluggedand the culture went into a semibatch/low dilution rate condition forabout 10 hours. The following morning, viscosity was up to 300 cp andwhen level and flow rates were corrected, it continued to increase. Forthe next 10 turnovers, specific productivity gradually increased to over0.35 gram of xanthan/gram cells/hour, and culture morphology becamenormal again. The culture was maintained at this high productivity forsome 30 turnovers after recovery without any evidence of culturedegeneration before being terminated by a pH upset. This highlyproductive culture was saved in a viable state and used to isolate thenovel Xanthomonas campestris strain referred to above.

After standard plate counts were made on the culture, using YM agar(Difco), 9 isolated colonies of varying sizes were picked andsubcultured on YM agar for purification. Three isolates were discardedas obvious duplicates, and the remaining six isolates were maintainedfor three bi-weekly transfers on YM agar slants. The isolates were thensubcultured to EMSY-1 broth and then maintained on EMSY-1 agar slants aswell as on the YM agar slants. In addition, cultures grown in EMSY-1broth were frozen in liquid nitrogen. The composition of this (EMSY-1)broth is given in Table II.

                  TABLE II                                                        ______________________________________                                        Component         Concentration (ppm)                                         ______________________________________                                        Glucose           10,000                                                      NH.sub.4 Cl       112 as N                                                    KH.sub.2 PO.sub.4 386 as P                                                    Na.sub.2 HPO.sub.4                                                                              390 as P                                                    MgSO.sub.4.7H.sub.2 O                                                                           40 as Mg                                                    CaCl.sub.2.2H.sub.2 O                                                                           10 as Ca                                                    NaCl              10 as Na                                                    FeCl.6H.sub.2 O   1 as Fe                                                     ZnSO.sub.4.7H.sub.2 O                                                                           0.33 as Zn                                                  MnSO.sub.4.H.sub.2 O                                                                            0.1 as Mn                                                   Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                   0.067 as Mo                                                 H.sub.3 BO.sub.3  0.033 as B                                                  KI                0.033 as I                                                  CuSO.sub.4.5H.sub.2 O                                                                           0.2 as Cu                                                   Citric Acid       500                                                         Yeast Extract     400                                                         ______________________________________                                    

The Xanthomonas campestris XCP-1 strain was obtained by pooling five ofthe six remaining isolates, i.e., P-101, P-102, P-104, P-105, and P-108,into one culture.

STRAIN DESCRIPTIONS

The above-mentioned strain of Xanthomonas campestris was characterizedwith respect to Xanthomonas campestris strain NRRL B-1459 from which itwas originally derived. The following descriptions characterize the newstrain:

I. Cell Morphology

1. After growth in EMSY-1 broth (composition in Table II) for 18 hoursat 28° C., cells appear singly and in pairs, with some chain formationapparent. Cell dimensions are 0.5-1.0 microns in width by 0.7 to 2microns in length. Chains of 5 to 30 microns in length may be formed.

2. After growth in YM broth (Difco) for 18 hours at 28° C., cells appearsingly and in pairs, with frequent chain formation apparent. Cells arelarger than in the minimal medium described above and have thedimensions of 0.75-1.2 microns in width by 1-4 microns in length. Chainsof 5-40 microns in length are found.

3. Strain XCP-1 does not exhibit motility, has a negative Gram stainreaction, and does not form endospores. These bacteria may be reproducedonly asexually by means of binary fission.

II. Colony Morphology

1. After 72 hours growth at 28° C. on EMSY-1 agar plates (composition inTable II) isolated colonies are yellow, circular, entire, mucoid,raised, and with a translucent periphery. Colony diameter is 2-3 mm.Colonies exhibit color variations in that both light yellow and darkeryellow colonies are present. This color variation is a stable propertyof the culture in that single isolated colonies of either color willgive rise to a mixture upon subculture.

2. After 72 hours growth on YM agar (Difco) plates at 28° C., isolatedcolonies are yellow, circular, entire, mucoid, and raised. Colonydiameter varies from 1.5 to 4 mm. Colonies are slightly darker yellowthan on EMSY-1 agar, but the variable color (light and dark yellow) isalso present.

3. After 72 hours growth at 28° C. on Nutrient Agar (Difco) platescontaining 1% dextrose, isolated colonies are circular, entire, mucoid,and raised. Colony diameter is 0.5-1.5 mm. Color variation is alsopresent and is of three types: (a) pale yellow to cream, (b) lightyellow, and (c) dark yellow.

III. Biochemical Characteristics

In order to determine whether strain XCP-1 is physiologically differentfrom Xanthomonas campestris NRRL B-1459, the following tests wereconducted.

1. Growth at 35° C. Inoculated slants of YM agar (Difco) and EMSY-1 agar(see Table II for composition) were incubated at 35° C. for five daysand results are given in Table III.

                  TABLE III*                                                      ______________________________________                                        Strain No.    YM Agar   EMSY-1 Agar                                           ______________________________________                                        XCP-1         2+        0                                                     B-1459        2+        0                                                     ______________________________________                                         *0 = no growth;                                                               1+ = slight growth;                                                           4+ = heavy growth                                                        

2. Growth Characteristics in Minimal Medium. Inoculated tubes of liquidEMS-2 medium (glucose, mineral salts, NH₄ Cl; see Example III forcomposition) were incubated at 28° C. for 96 hours. Strain XCP-1 showedheavy growth throughout the tubes with a ragged surface pellicle andclumping in the broth. Strain B-1459 showed less growth overall and onlya slight surface growth.

3. Hydrolysis of Gelatin, Casein, and Starch. Solid agar mediaindividually containing 0.4% gelatin, 0.4% casein, or 0.3% solublestarch were prepared and used according to the procedure in"Identification Methods for Microbiologists", 1966, B. M. Gibbs and F.A. Skinner, eds., Academic Press, p. 12.

                  TABLE IV                                                        ______________________________________                                        Strain No. Gelatin      Casein  Starch                                        ______________________________________                                        XCP-1      4+           4+      4+                                            B-1459     4+           4+      4+                                            ______________________________________                                    

Strains B-1459 and XCP-1 showed complete hydrolysis of all threesubstrates as shown in Table IV.

4. Action on Litmus Milk. Cultures inoculated into Litmus Milk medium(Difco) were incubated at 28° C. for three weeks, according to themethod of Ivanoff et al. (1938, J. Bacteriol, 35 235). Strains XCP-1 andB-1459 were both active in litmus milk with peptonization, litmusreduction, and precipitate formation.

5. Hydrogen Sulfide Production. The medium for H₂ S production wasprepared according to the method of Hayward and Hotchkiss (1961, J. Gen.Microbiol. 26, 133-140). H₂ S production was determined by the use oflead acetate paper strips suspended over the medium in loosely cappedtubes. The cultures were incubated for six days at 28° C. and observedfor blackening of the strips. Each of the strains produced hydrogensulfide.

6. Urease Production. Urea medium was prepared according to the methodof Christensen (1946, J. Bacteriol. 52 461-466). The slants wereinoculated and incubated at 28° C. for 14 days. A red to violet color inthe medium would be indicative of urea hydrolysis. Urease production wasfound to be negative for each of the strains tested.

7. Growth in Presence of Salt. Basal media containing NaCl atconcentrations of 1, 2, 3, 4, and 5% were prepared according to themethod of Hayward and Hotchkiss (1961. J. Gen. Microbiol. 26 133-140).Cultures were inoculated and incubated at 28° C. for 14 days. Bothstrains tested gave an identical growth pattern as shown in Table V.

                  TABLE V                                                         ______________________________________                                        Salt Conc'n                                                                   Strain No.                                                                              1%        2%     3%     4%   5%                                     ______________________________________                                        XCP-1     4+        3+     3+     2+   0                                      B-1459    4+        3+     3+     2+   0                                      ______________________________________                                    

8. Carbohydrate Assimilation Pattern. A basal carbohydrate assimilationmedium was prepared according to the method of Hayward and Hotchkiss(1961. J. Gen. Microbiol. 26 133-140). Each strain was inoculated intoreplicate tubes containing the carbohydrates shown in Table VI, andincubated for 14 days at 28° C.

                  TABLE VI                                                        ______________________________________                                        Carbohydrate     XCP-1   B-1459                                               ______________________________________                                        Glucose          +       +                                                    Galactose        +       +                                                    Arabinose        +       +                                                    Mannose          +       +                                                    Melibiose        +       +                                                    Cellobiose       +       +                                                    Sucrose          weak    weak                                                 Fructose         weak    weak                                                 Trehalose        +       +                                                    Xylose           -       -                                                    Mannitol         +       +                                                    Lactose          -       -                                                    Maltose          +       +                                                    ______________________________________                                    

As can be seen in Table VI, each strain gave an identical assimilationprofile.

9. Oxidase Production. Using isolated colonies from 72 hour old YM agar(Difco) plates, the strains were tested for presence of indophenoloxidase using the method of Gaby and Hadley (1957. J. Bacteriol. 74356-358). Each strain was positive for oxidase.

10. Catalase Production. Growth from a 48 hour YM agar (Difco) slant wastested for catalase activity by emulsifying a loopful of culture in adrop of 3% H₂ O₂ and observing for effervescence. Strains XCP-1 andB-1459 were both weakly positive.

11. Utilization of Organic Acids. EMS-2 basal medium without glucose(shown in Table VII) was prepared.

                  TABLE VII                                                       ______________________________________                                        Component         Concentration (ppm)                                         ______________________________________                                        Glucose           22,500                                                      NH.sub.4 Cl       224 as N                                                    KH.sub.2 PO.sub.4 150 as P                                                    MgSO.sub.4.7H.sub.2 O                                                                           40 as Mg                                                    CaCl.sub.2.2H.sub.2 O                                                                           10 as Ca                                                    Citric Acid       500                                                         FeCl.sub.3.6H.sub.2 O                                                                           2 as Fe                                                     ZnSO.sub.4.7H.sub.2 O                                                                           0.66 as Zn                                                  CuSO.sub.4.5H.sub.2 O                                                                           0.4 as Cu                                                   MnSO.sub.4.H.sub.2 O                                                                            0.2 as Mn                                                   Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                   0.13 as Mo                                                  H.sub.3 BO.sub.3  0.066 as B                                                  KI                0.066 as I                                                  NaCl              10 as Na                                                    ______________________________________                                    

Replicate tubes containing 1% citric, malic, succinic, benzoic, andtartaric acids were inoculated and incubated at 28° C. for 14 days andobserved for extent of growth. As shown by the result given in TableVIII, both strains were identical.

                  TABLE VIII                                                      ______________________________________                                        Organic Acid     XCP-1   B-1459                                               ______________________________________                                        Citrate          3+      3+                                                   Malate           4+      4+                                                   Succinate        4+      4+                                                   Benzoate         0       0                                                    Tartrate         1+      1+                                                   ______________________________________                                    

12. Indole Production. The strains were tested for indole production forthe same peptone-water medium used to test for H₂ S production,following the method of Hayward and Hotchkiss (1961. J. Gen. Microbiol.26 133-140). All strains were negative for indole production.

13. Acetoin Production. The strains were tested for acetoin productionusing MRVP medium (Difco) after incubation of the inoculated culturesfor six days at 28° C., following the method given in the reference in(12) above. None of the strains tested positive for acetoin.

Summary of Characterization Studies

Strain XCP-1 is essentially indistinguishable from X. campestris strainNRRL B-1459 on the basis of cell morphology. However, definitedifferences in colonial morphology make this strain distinguishable fromB-1459. Strain XCP-1 exhibits color variations; i.e., light yellow(larger, 3-4 mm) and darker yellow (smaller, 2-3 mm) colonies on EMSY-1and YM agar plates, when compared to B-1459. XCP-1 also produces smallercolonies than B-1459 on Nutrient Glucose Agar.

In terms of physiological characteristics, this strain is very similarto NRRL B-1459 except that it grows better on a minimal medium thanB-1459.

The foregoing is intended to point out that, while the majordistinguishing characteristics of the XCP-1 strain lie in its highxanthan specific productivity and resistance to degeneration incontinuous culture, other distinguishing characteristics neverthelessare present.

In carrying out the process of the present invention, the fermentermedium is seeded with an inoculum of culture grown in the same medium asthat to be used for fermentation at an inoculum level of 5-10% of themedium volume. The culture is grown in a batch mode for 24-48 hours,until a desired cell concentration is reached (usually 1.5-2.5 gramcells/liter). Thereafter, continuous flow of medium is started into thefermenter such that the dilution rate is 75% or less of the specificgrowth rate at which the organism is growing at that point. Continuousharvesting of a volume of culture broth equal to the volume of mediumintroduced is also carried out. After approximately two cultureturnovers, the dilution rate is adjusted as desired. Xanthan gum, whichexists in the recovered broth, can be used without further purification,or filtered to remove cells, or can be precipitated with an alcohol,such as ethyl or isopropyl alcohol, with or without initial cellremoval. The medium used in this process is preferably a minimal mediumconsisting primarily of inorganic salts, NH₄ Cl, glucose, and citricacid, with or without additional yeast extract or yeast autolysate.

The term "minimal medium" as used throughout the present description andclaims should be interpreted to cover media of the type generallyreferred to herein and specifically in the Examples, together withmodifications apparent to those skilled in this field.

Operating conditions to be employed in the process of our inventioninclude the following:

Agitation: 100-2000 rpm

Preferably: 500-1000 rpm

Air Rate: 0.1-2 vol./vol./min.

Preferably: 0.5-1 vol./vol./min.

Temperature: 20°-35° C.

Preferably: 25°-30° C.

pH: 5-8

Preferably: 6.4-7.4

Dissolved Oxygen: 10-90% saturation

Preferably: 20-60% saturation

Dilution Rate: 0.01-0.15 hr⁻¹

Preferably: 0.04-0.1 hr⁻¹

Our invention will be illustrated by reference to the following specificexamples:

EXAMPLE I

This Example shows that when Xanthomonas campestris NRRL B-1459, isgrown in a minimal medium in continuous culture, the organism exhibitsonly low specific productivity and degenerates in a short time. Theculture was grown in a 28 liter fermenter in a minimal medium having acomposition shown in Table IX.

                  TABLE IX                                                        ______________________________________                                        Component        Concentration (ppm)                                          ______________________________________                                        Glucose          22,000                                                       NH.sub.4 Cl      300 as N                                                     KOH              1,000 as K                                                   H.sub.3 PO.sub.4 150 as P                                                     MgSO.sub.4       40 as Mg                                                     CaCl.sub.2       10 as Ca                                                     NaCl             10 as Na                                                     Citric Acid      500                                                          FeSO.sub.4       3 as Fe                                                      ZnSO.sub.4       1 as Zn                                                      MnSO.sub.4       0.3 as Mn                                                    Na.sub.2 MoO.sub.4                                                                             0.2 as Mo                                                    H.sub.3 BO.sub.3 0.1 as B                                                     KI               0.1 as I                                                     CuSO.sub.4       0.6 as Cu                                                    ______________________________________                                    

Xanthomonas campestris B-1459 was maintained on YM agar (Difco) slantsat 4° C. and transferred to fresh agar slants at bi-weekly intervals.For inoculum preparation, a loopful of culture from a fresh (<3 day old)slant was inoculated into a 16×125 mm tube containing 7 ml of YM broth.The culture was incubated at 28° C. on a rotary shaker at 150 rpm, at a20° inclination for 18 hours. At this point, the contents of the tubewere transferred to 50 ml YM broth in a 500 ml Erlenmeyer flask, andincubated at 28° C. on a rotary shaker at 250 RPM for 18-24 hours. Next,the contents of the flask were transferred to a 2000 ml Fernbach flaskcontaining 700 ml of mineral salt-glucose-NH₄ Cl medium, of thecomposition given in Table IX. This was incubated under the sameconditions as for the 50 ml flask, but for a total of 40 hours. Next,the entire culture was used to inoculate 20 liters of the same mediumcontained in a 28 liter New Brunswick fermenter (Model CMF-128S). Theinitial operating conditions employed were as follows:

Temperature: 29° C.

pH: 6.0

Agitation: 230 rpm

Air Rate: 0.2-0.4 vol/vol/min

Dissolved O₂ : 90% saturation

After an initial growth lag of about 30 hours, cell growth proceededover the next 30 hours. When the cell concentration reached 0.9gm/liter, continuous operation was started at an initial dilution rateof 0.07 hr⁻¹. Within 48 hours, the cell concentration rose to 2.5gm/liter. After about 10 culture turnovers, the viscosity and specificproductivity started to decline and were eventually almost totally lost.Cell morphology became abnormal and gum quality deteriorated badly. Allof these changes proved to be irreversible and the culture did notrevert to normal. The results obtained in this run are given in Table X.

                                      TABLE X                                     __________________________________________________________________________                        Xanthan                                                                              Xanthan                                            Time Cell Xanthan   Volumetric                                                                           Specific       Total                               Period                                                                             Conc'n                                                                             Conc'n                                                                             Viscosity                                                                          Productivity                                                                         Productivity                                                                           Dilution                                                                            Culture                             (Hrs)                                                                              (gm/l)                                                                             (%)  (cp) gm/l/hr)                                                                             (gm/gm cells/hr)                                                                       Rate (hr.sup.-1)                                                                    Turnovers                           __________________________________________________________________________     0-34                                                                              1.3-1.9                                                                            .26-.30                                                                            120-340                                                                            .20    .11-.15  .07-.08                                                                             0-2.5                                34-130                                                                            2.6-2.56                                                                           .32-.39                                                                            640-850                                                                            .23-.31                                                                              .10-.13   .07-.085                                                                           2.5-9.7                             130-178                                                                            1.7-2.17                                                                            .23-.265                                                                          420-430                                                                            .17-.22                                                                              .09-.10  .07-.08                                                                             9.7-13.7                            178-202                                                                            1.0  .187 160  .13    .13      .07   13.5-15                             202-266                                                                            .6-1.1                                                                             .12-.14                                                                            28-48                                                                               .1-.11                                                                               .09-.175                                                                              .077  15-20.1                             __________________________________________________________________________

EXAMPLE II

Xanthomonas campestris XCP-1, ATCC 31600, after isolation as describedabove, was maintained on EMSY-1 agar slants. These slants were stored at4° C. and the culture was transferred to fresh slants at bi-weeklyintervals.

For inoculum preparation, a loopful of culture from a fresh (<3 day old)slant was inoculated into a 16×125 ml tube containing 7 ml of EMSY-1broth. The culture was incubated at 28° C. on a rotary shaker at 150 rpmat a 20° C. inclination for 18 hours. At this point, the contents of thetube were transferred to 50 ml of EMS-1 broth (which has the samecomposition as the EMSY-1 broth except no yeast extract is present) in a500 ml Erlenmeyer flask, which was incubated at 28° C. on a rotaryshaker at 250 rpm for 18 to 24 hours. Next 10 ml volumes of the culturewere inoculated into each of two 1000 ml Erlenmeyer flasks containing100 ml of EMS-1 medium. These were incubated at 28° C. on a rotaryshaker at 250 rpm for 18 to 24 hours. The culture contents of bothflasks were combined and 150 ml of the culture was used to seed 3000 mlof EMS-3 medium (shown in Table XI) contained in a 7.5 liter fermenter(New Brunswick Model MF-107).

                  TABLE XI                                                        ______________________________________                                        Component       Concentration (Per Liter)                                     ______________________________________                                        NH.sub.4 Cl     1.28 gm                                                       KH.sub.2 PO.sub.4                                                                             0.66 gm                                                       MgSO.sub.4.7H.sub.2 O                                                                         0.41 gm                                                       CaCl.sub.2.2H.sub.2 O                                                                         0.04 gm                                                       NaCl            0.026 gm                                                      Citric Acid     0.5 gm                                                        FeCl.sub.3.6H.sub.2 O                                                                         15 mg                                                         ZnSO.sub.4      2.5 mg                                                        MnSO.sub.4      0.82 mg                                                       CuSO.sub.4      1.5 mg                                                        Na.sub.2 MoO.sub.4                                                                            0.43 mg                                                       H.sub.3 BO.sub.3                                                                              0.57 mg                                                       KI              0.13 mg                                                       Glucose         22.6 gm                                                       ______________________________________                                    

The fermentation was carried out under the following initial operatingconditions:

Temperature: 28° C.

pH: 7.0

Agitation: 300 rpm

Air Rate: 0.5 vol/vol min/min

Dissolved O₂ : 90% of saturation

After 41 hours during which time cell growth proceeded in a batch mode,the agitation rate was increased to 400 rpm and the air rate to 1vol/vol/min. After an additional 12 hours of growth at which point thecell concentration was about 1.6 gm/liter, continuous operation wasstarted at an initial dilution rate of 0.06 hr⁻¹ using the same medium(EMS-3) referred to above. After 15 hours of continuous operation, theagitation rate was increased to 600 rpm due to increasing viscosity inthe culture. After correcting a pH upset which caused the pH to rise to8.0, specific productivity increased from 0.26 to 0.29 gm/gm cells/hour.These results are summarized in Table XII.

                                      TABLE XII                                   __________________________________________________________________________                         Xanthan                                                                              Xanthan                                           Time Cell Xanthan    Volumetric                                                                           Specific       Total                              Period                                                                             Conc'n                                                                             Conc'n                                                                             Viscosity                                                                           Productivity                                                                         Productivity                                                                           Dilution                                                                            Culture                            (Hrs)                                                                              (gm/l)                                                                             (%)  (cp)  gm/l/hr)                                                                             (gm/gm cells/hr)                                                                       Rate (hr.sup.-1)                                                                    Turnovers                          __________________________________________________________________________     0-36                                                                              1.58 0.56 830-940                                                                             0.41-.42                                                                             .26      .07-.073                                                                              0-2.5                            pH reduced from 8 to 6.8                                                       36-114                                                                            1.48-1.67                                                                          .52-.65                                                                             860-1720                                                                           .38-.45                                                                              .23-.29  .07-.075                                                                            2.5-8.5                            115-131                                                                            1.46  .50 1040  .39    .27      .078  8.5-9.6                            132-160                                                                             .48-1.17                                                                          .18-.47                                                                            120-550                                                                             .13-.38                                                                              .27-.33  .07-.073                                                                             9.6-11.7                               cell concentration decreased                                             160-188                                                                            batch growth                                                             188-206                                                                            1.09-1.26                                                                          .47-.49                                                                            410-870                                                                             .24-.29                                                                              .19-.27  .05-.06                                                                             11.7-12.2                          206-233                                                                            batch growth                                                             233-325                                                                            1.69-1.86                                                                          .68-.80                                                                            1780-2310                                                                           .30-.42                                                                              .18-.25  .04-.055                                                                            12.2-16.8                          325-379                                                                            1.55-1.71                                                                          .53-.64                                                                             900-1440                                                                           .38-.46                                                                              .25-.27  .07-.073                                                                            16.8-20.7                          397-398                                                                            dilution rate upset                                                      398-517                                                                            1.67-1.97                                                                          .44-.53                                                                            500-870                                                                             .32-.38                                                                              .18-.20  .073-.075                                                                           20.7-30.4                          __________________________________________________________________________

At 5.5 culture turnovers, the specific productivity started to declineto 0.24-0.26 due to an increase in dilution rate and temperature and pHcycling. After two periods of batch growth made in attempts to bring thecell concentration back to normal, the culture was maintained at aspecific productivity of 0.2 gm/gm cells per hour, which was stillhigher than that exhibited by the parent strain (B-1459). Specificproductivity then improved to 0.22-0.23 gm/gm cells per hour at adilution rate of 0.05 hr⁻¹, and increased again to 0.26-0.27 when thedilution rate was increased to 0.07 hr⁻¹. At 395 hours, the specificproductivity slowly declined to 0.18-0.19 gm/gm cells per hour, whichwas maintained until 517 hours. Thus, Xanthomonas campestris XCP-1 ATCC31600, was grown continuously for a period of 30 culture turnovers atspecific productivities of 0.18-0.19 up to 0.29 gm/gm cells/hour whichis 1.5 to 2.4 times higher than the specific productivity of Xanthomonascampestris NRRL B-1459, and culture degeneration of any consequence wasavoided.

EXAMPLE III

In this example, an inoculum of Xanthomonas campestris XCP-1, ATCC31600, was prepared in accordance with the procedure outlined in ExampleII except that the 50 ml and 100 ml cultures were grown in EMS-2 medium(shown in Table VII) which contained 0.04 gm/liter of yeast extract.After 24 hours of batch growth, the cell concentration was about 1.5gm/liter and continuous operation was started at a dilution rate of0.059 hr⁻¹. The agitation rate was increased at this point to 800 rpm.

The results of this run are given below in Table XIII.

                                      TABLE XIII                                  __________________________________________________________________________                         Xanthan                                                                              Xanthan                                           Time Cell Xanthan    Volumetric                                                                           Specific       Total                              Period                                                                             Conc'n                                                                             Conc'n                                                                             Viscosity                                                                           Productivity                                                                         Productivity                                                                           Dilution                                                                            Culture                            (Hrs)                                                                              (gm/l)                                                                             (%)  (cp)  gm/l/hr)                                                                             (gm/gm cells/hr)                                                                       Rate (hr.sup.-1)                                                                    Turnovers                          __________________________________________________________________________     0-21                                                                              1.9-2.2                                                                             .8-.89                                                                            2450   .47-.475                                                                            .22-.24  .059    0-0.8                            21-95                                                                              1.75-2.0                                                                           .57-.66                                                                            1280-1720                                                                           .48-.55                                                                              .25-.30   .08-.088                                                                           0.8-5.8                             95-265                                                                             2.1-2.35                                                                          .61-.72                                                                            1418-1750                                                                           .49-.58                                                                              .22-.25  .076-.084                                                                           19.2-28.4                          __________________________________________________________________________

The data show that Xanthomonas campestris XCP-1 ATCC 31600 was grown for19 culture turnovers at a high specific productivity ranging from 0.22to 0.30 gm xanthan/gm cells/hr. Only after 265 hours of continuousoperation when the unit became contaminated with an unknown bacteriumdid the specific productivity decrease, ranging from 0.16 to 0.25 whichis still substantially higher than the specific productivity of theparent strain B-1459. Thus, it is seen that Xanthomonas campestris XCP-1ATCC 31600 can also be grown in a minimal medium supplemented with 0.04%yeast extract. Actually, any amount of yeast extract or yeast autolysateranging from 0.005% to 0.1% can be used although 0.02% to 0.04% isoptimal when balancing utility and cost considerations.

We claim:
 1. A method for the production of a heteropolysaccharide which comprises continuously culturing a degenerative resistant strain of bacteria designated Xanthomonas campestris XCP-1, having the identifying characteristics of ATCC 31600, in an aqueous medium comprising the assimilable sources of carbon, nitrogen and inorganic substances wherein said medium is continuously fed to a fermentation zone to produce said polysaccharide, and withdrawing the resulting fermented medium from said zone.
 2. The method of claim 1 in which said medium is a minimal medium.
 3. The method of claim 2 wherein said minimal medium also contains as a complex nitrogen source, one of the group consisting of yeast extract and yeast autolysate.
 4. The method of claim 1 in which the fermentation is conducted at a specific xanthan productivity in excess of 0.2 gm xanthan/gm cells/hr.
 5. The method of claim 1 wherein said fermented medium is withdrawn from said zone at a rate such that an essentially steady state condition is maintained in said zone.
 6. A method for the production of a heteropolysaccharide which comprises continuously culturing a degenerative resistant strain of Xanthomonas campestris P-1 having the identifying characteristics of ATCC 31600 in a minimal medium containing a growth limiting nutrient and wherein said medium is continuously fed to a first fermentation zone to produce additional amounts of said strain together with said heteropolysaccharide, thereafter transferring the effluent from said first zone to a second fermentation zone, and adding a fermentable sugar to said effluent in said second zone whereby the formation of heteropolysaccharide in said second zone is maximized.
 7. The method of claim 6 in which the fermentable sugar employed in glucose.
 8. The method of claims 1 or 6 in which the heteropolysaccharide is xanthan.
 9. The biologically pure culture of a novel strain of Xanthomonas campestris XCP-1, having the identifying characteristics of ATCC 31600, said strain being capable of producing xanthan gum in recoverable amounts upon fermentation of an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances and being resistant to cell degeneration when subjected to continuous fermentation conditions.
 10. A biologically pure culture consisting essentially of Xanthomonas campestris XCP-1 ATTC
 31600. 11. A biologically pure culture consisting essentially of Xanthomonas campestris XCP-1 ATCC 31600, said culture being degenerative resistant when continuously cultured in an aqueous nutrient medium, and capable of producing xanthan gum in good yields.
 12. A bacterial culture consisting essentially of Xanthomonas campestris XCP-1 ATTC
 31600. 13. A bacterial culture consisting essentially of Xanthomonas campestris XCP-1 ATTC 31600, said culture being degenerative resistant when continuously cultured in an aqueous nutrient medium, and capable of producing xanthan gum in good yields.
 14. A bacterial culture consisting essentially of Xanthomonas campestris XCP-1 ATTC 31600, said culture capable of producing xanthan gum in recoverable amounts upon fermentation of an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic substances. 